Pharmaceutical formulations and uses thereof in the treatment of female sexual dysfunction

ABSTRACT

The present invention relates to the use of a combination of a PDE5-inhibitor and testosterone for the preparation of a medicament for the treatment of Female Sexual Dysfunction.

CROSS-REFERENCE TO RELATED APPLICATIONS

This application is a continuation of PCT International PatentApplication No. PCT/NL2005/000355, filed on May 11, 2005, designatingthe United States of America, and published, in English, as PCTInternational Publication No. WO 2005/107810 A2 on Nov. 17, 2005, whichapplication claims priority to European Patent Application Serial No.04078455.5 filed Dec. 21, 2004, which in turn claims priority toEuropean Patent Application Serial No. 04078381.3 filed Dec. 13, 2004,which in turn claims priority to European Patent Application Serial No.04078380.5 filed Dec. 13, 2004, which in turn claims priority toEuropean Patent Application Serial No. 04078033.0 filed Nov. 4, 2004,and which in turn claims priority to European Patent Application SerialNo. 04076402.9 filed May 11, 2004, the contents of the entirety of eachof which is hereby incorporated herein by this reference.

TECHNICAL FIELD

The invention relates to the field of female sexual dysfunction. Itspecifically relates to the influence of the combination oftestosterone, or an analogue thereof, and a PDE5 inhibitor (such assildenafil, vardenafil or tadalafil) on sexual health in female subjectswith Female Sexual Dysfunction (such as Female Sexual Arousal Disorder(FSAD) or Female Sexual Desire Disorder (FSDD)).

BACKGROUND

Female Sexual Dysfunction (FSD) refers to various disturbances orimpairments of sexual function, including a lack of interest in sexualactivity, repeated failure to attain or maintain sexual excitement, andinability to attain an orgasm following sufficient arousal. A recentstudy estimated that 43% of women suffer from sexual dysfunction in theUSA.¹ Low sexual desire (22% prevalence) and sexual arousal problems(14% prevalence) belong to the most common categories of sexualdysfunction of women. These categories are convenient in providingworking definitions and an accepted lexicon for researchers andtherapists. However, it may be incorrect to assume that these disordersare fully independent of each other. Both case studies andepidemiological studies demonstrate that these disorders can overlap andmay be interdependent. In some cases, it may be possible to identify theprimary disorder that led to the others, but in many cases, this may beimpossible.

For the treatment of female sexual disorder, a number of differenttreatments with greater or lesser degrees of success have been suggestedand applied. These treatments have either not been completely successfulor the side effects are hardly acceptable. The present inventionprovides a new combination of therapeutic substances that is effectiveand does not have serious side effects when given in a particular dosagescheme.

DISCLOSURE OF THE INVENTION

Thus, the invention provides the use of a combination of a PDE5inhibitor and testosterone, or an analogue thereof, in the preparationof a medicament for the treatment of female sexual dysfunction.

According to the invention, although it is not considered bound bytheory, an effect on the central nervous system and the peripheralsystem are required, whereby the signal to the central system isprovided by testosterone, or an analogue thereof (having the same kindof activity), and the peripheral signal is provided by a PDE5 inhibitor.

According to the invention, the level of free testosterone should be apeak plasma level of free testosterone of at least about 0.010 nmol/L,which will typically occur about 20 minutes after administration of thetestosterone. According to the invention, the effect of the peak plasmalevel of at least 0.010 nmol/l of free testosterone is to be reached atabout the same time as the effect of the PDE5 inhibitor. For an optimaleffect, it is desired that the peak effect of both compounds coincide.However, even if the peak effects only partly overlap, this stillresults in the desired effect (treatment of FSD). There is a time lagfor the effect of testosterone (or the analogue) of about three to sixhours (more specifically, around three to 4.5 hours), in particular,around four hours. PDE5 inhibitors, such as vardenafil and sildenafil,typically reach their peak plasma concentration (which should be atleast 35 ng/ml for sildenafil, 2 μg/L for vardenafil and 40 μg/L fortadalafil) after about one hour after administration and thus, the twopharmaceuticals are preferably presented as a kit of parts withinstructions about the administration, or are packaged in one capsule orformula with differential release properties for the two compounds.

Testosterone in the circulation is typically bound by SHBG (steroidhormone binding globulin) and by albumin. It is important that the peakplasma level of testosterone as defined in the present invention ispresent and calculated as free testosterone, so a fraction is not boundby albumin and SHBG. Thus, the dose of testosterone given should be highenough to saturate the albumin and SHBG (i.e., the concentration oftestosterone must be high enough to overcome complete binding oftestosterone by SHBG or albumin) or another way of avoiding binding toalbumin or SHBG must be designed, such as the use of a competitor forthe testosterone binding site on SHBG.

Testosterone is preferably given in a formulation wherein there is ashort high peak in the blood circulation of the subject to which it isadministered. The invention, therefore, provides a use, wherein thetestosterone, or an analogue thereof, is provided in the form of asublingual formulation, preferably a sublingual formulation comprising acyclodextrin as a carrier. A typical example of such a formulation isgiven in hydroxypropyl-beta cyclodextrin, but other beta cyclodextrinsand other usual excipients, diluents and the like are within the skillof the art for preparing a formulation comprising testosterone, or ananalogue thereof, which releases essentially all of the testosteronewithin one short burst. That burst will typically be within a short timeinterval (for example, within 60 to 120 seconds, more preferably within60 seconds) upon administration, leading to blood peak levels oftestosterone about 15 to 20 minutes later. In a preferred embodiment,the pharmaceutical is designed for sublingual administration and evenmore preferred, the composition comprises cyclodextrin such ashydroxypropyl-beta cyclodextrin. A typical example of a preparedtestosterone sample (for 0.5 mg of testosterone) consists of 0.5 mgtestosterone, 5 mg hydroxypropyl-beta cyclodextrin (carrier), 5 mgethanol, and 5 ml water, but each of the amounts of these substancesmight be higher or lower.

Of course, the pharmaceutical preparation comprising a PDE5 inhibitorshould also be designed to give a peak plasma level at about the timewhen the testosterone effect is maximal. Such compositions are withinthe skill of the art. A typical example for oral administration is givenin vardenafil HCl, which is designated chemically as piperazine,1-[[3-(1,4-dihydro-5-methyl-4-oxo-7-propylimidazo[5,1-f][1,2,4]triazin-2-yl)-4-ethoxyphenyl]sulfonyl]-4-ethyl-,monohydrochloride. In addition to the active ingredient, vardenafil HCl,each tablet contains microcrystalline cellulose, crospovidone, colloidalsilicon dioxide, magnesium stearate, hypromellose, polyethylene glycol,titanium dioxide, yellow ferric oxide, and red ferric oxide. Anotherexample is given in sildenafil citrate, which is chemically designatedas1-[[3-(6,7-dihydro-1-methyl-7-oxo-3-propyl-1Hpyrazolo[4,3-d]pyrimidin-5-yl)-4-ethoxyphenyl]sulfonyl]-4-methylpiperazinecitrate. In addition to the active ingredient, sildenafil citrate, eachtablet contains the following ingredients: microcrystalline cellulose,anhydrous dibasic calcium phosphate, croscarmellose sodium, magnesiumstearate, hydroxypropyl methylcellulose, titanium dioxide, lactose,triacetin, and FD & C Blue #2 aluminum lake. Another example is given intadalafil, which is chemically designated aspyrazino[1′,2′:1,6]pyrido[3,4-b]indole-1,4-dione,6-(1,3-benzodioxol-5-yl)-2,3,6,7,12,12a-hexahydro-2-methyl-, (6R,12aR)-.In addition to the active ingredient, tadalafil, each tablet containsthe following ingredients: croscarmellose sodium, hydroxypropylcellulose, hypromellose, iron oxide, lactose monohydrate, magnesiumstearate, microcrystalline cellulose, sodium lauryl sulfate, talc,titanium dioxide, and triacetin.

It is clear, that preferably the (peak) effect of PDE5 inhibitor, aswell as the (peak) effect of testosterone, coincide (completely). It is,however, noted that if the peak effect of testosterone and of the PDE5inhibitor only partly overlap, this still results in the desired effect.When the testosterone is provided such that it essentially releases allof the testosterone within one short burst to a female subject, the PDE5inhibitor is preferably provided such that it results in a peak plasmaconcentration at least three hours after the administration oftestosterone. Even more preferred, the PDE5 inhibitor effect is present3.5 to 5.5 hours after the intake of testosterone. It is clear that theexact time of PDE5 inhibitor administration is dependent on the type offormulation used. If the PDE5 inhibitor formulation is released shortlyafter administration, it is of no use to provide it at the same time asthe testosterone is provided, because there will be hardly any overlapof effect. If it takes some time before the PDE5 inhibitor is availablefrom the used formulation, for example, three to four hours, it canbe/is administrated at the same time the testosterone is administrated.

For the present invention, the routes of administration of choice arethose that are the least invasive. Motivation for sexual behavior shouldnot be negatively influenced by invasive routes of administration.Because there is a time lag in the effect of testosterone, the two drugsnecessary for a central effect and a peripheral effect cannot beadministered at the same time (unless the administration of the PDE5inhibitor is designed such that the drug is released after 3.5 to 5.5hours after administration).

The invention, therefore, provides a kit of parts comprising at leastone pharmaceutical composition comprising testosterone, or an analoguethereof, and at least one pharmaceutical composition comprising a PDE5inhibitor, whereby the composition comprising testosterone is designedto release all testosterone essentially immediately (for example, within60 seconds) at the target site. The kit preferably contains instructionsto use a pharmaceutical composition comprising testosterone 3.5 to 5.5hours prior to sexual activity and a pharmaceutical compositioncomprising a PDE5 inhibitor one to two hours prior to sexual activity.

The kit of parts may comprise a sublingual formulation of testosterone,or an analogue thereof, and a tablet or another formulation comprising aPDE5 inhibitor. The preferred PDE5 inhibitors are sildenafil, vardenafilor tadalafil. The amount of testosterone per pharmaceutical compositioncomprising testosterone is at least 0.3 mg testosterone and at most 2.5mg testosterone. Higher or lower doses may be necessary depending on thealbumin and SHBG levels and the weight of the subject to be treated. Thepharmaceutical composition comprising a PDE5 inhibitor comprises atleast 25 mg sildenafil (or 5 mg vardenafil, or 5 mg tadalafil) and atmost 100 mg sildenafil (or 20 mg vardenafil, or 20 mg tadalafil), orcomparable dosages of other PDE5 inhibitors. Again, these doses may varywith the weight of the patient.

For the reasons already outlined above, a kit according to the inventionmay further comprise a compound capable of competing with testosterone,or an analogue thereof, for SHBG binding.

In a preferred embodiment, the testosterone analogue is a precursor ormetabolite of testosterone. In case a precursor of testosterone is used,the kit further comprises instructions (if necessary) to increase thetime period of 3.5 to 5.5 hours by adding the time that is needed toconvert the precursor into testosterone. In case a metabolite oftestosterone is used, the time period of 3.5 to 5.5 hours is shortened.

In order to further enhance the effects of the kit of parts of theinvention, the kit may further comprise means for cognitiveinterventions and stimulation. Such information may be present on anydata carrier (paper, CD, DVD), passive or interactive, or it may be alink to a website at least partially designed for the purpose ofcognitive stimulation. Sometimes it is preferred to present thecognitive stimulatory information subconsciously, e.g., subliminally.

To further enhance the effects of the kit of the present invention, asubstance may be added to the kit that stimulates the mesolimbicdopaminergic pathway in the subject. This pathway is concerned with arelatively different kind of reward system that helps provide anincrease in reward-seeking involved in sexual behavior. Examples of suchcompounds are Apomorphine, a dopamine D2 agonist; Aripiprazol, a partialdopamine D2 agonist; Pergolide, a nonselective dopamine (DA) agonist;Pramipexole, a new dopamine receptor agonist with preference for D3compared to D2 and D4 receptors; Bromocriptine, a nonselective dopamine(DA) agonist; Ropinirole hydrochloride, a non-ergoline dopamine agonistwith a relatively high in vitro specificity and full intrinsic activityat the D₂ and D₃ dopamine receptor subtypes that binds with higheraffinity to D₃ than to D₂ or D₄ receptor subtypes; Roxindole, a potent(autoreceptor)-“selective” D3 dopamine agonist; Cabergoline, a dopamineD2 agonist; Lisuride, a nonselective dopamine (DA) agonist, and theautoreceptor antagonists, (+)-AJ 76, a D3-preferring, dopamine (DA)autoreceptor antagonist; (+)-UH232, a stimulant of dopaminergictransmission, which may preferentially antagonize autoreceptors ofdopamine nerve terminals, as well as the reuptake blockers; Bupropion,an inhibitor of the neuronal uptake of norepinephrine, serotonin anddopamine; Amineptine, a (relatively) selective dopamine reuptakeinhibitor; GBR 12909 (vanoxerine), a dopamine reuptake inhibitor; andAmantadine, a NMDA receptor antagonist and dopamine reuptake inhibitor.

To further enhance the effects of the kit of the present invention, asubstance is (optionally) added that inhibits the central and peripheraladrenergic tone, i.e., inhibits or dampens central and peripheralextracellular norepinephrine concentrations. Activation of alpha 2receptors located in the central nervous system results in inhibition ofsympathetic tone. Examples of such compounds are clonidine, an alpha 2agonist; imidazoline, a partial alpha 2 agonist; and dexmedetomidine, analpha 2 agonist.

The kit of parts is useful for any individual suffering from any form ofFSD, be it through psychological or physiological causes or combinationsthereof. It is thus also useful for subjects having FSD because of othermedicines and/or drugs (such as SSRIs) and subjects suffering fromhypogonadism, etc.

DESCRIPTION OF THE DRAWINGS

FIG. 1 consists of graphs depicting a delay between testosteroneadministration and VPA in healthy subjects.

FIG. 2 illustrates the results of VPA measurement in women sufferingfrom FSD receiving a placebo, combined with a PDE5 inhibitor or thepharmaceutical preparation according to the present invention comprisingtestosterone and a PDE5 inhibitor (this last combination is also called“Lybrido”).

FIG. 3 illustrates the results of VPA measurement in women sufferingfrom FSD receiving a placebo, and PDE5 inhibitor, testosterone or thepharmaceutical preparation according to the present invention.

FIG. 4 shows VPA measurements of different treatments.

FIG. 5 illustrates results relating to sexual abuse, attentional biasand VPA.

DETAILED DESCRIPTION OF THE INVENTION

Low sexual desire, sexual arousal problems and hampered orgasm arecandidates for psychopharmacological treatment. These categories ofsexual problems are also linked to three (transitional and overlapping)phases of the human sexual response (sexual desire, sexual arousal andorgasm), which are regulated by relatively independent neurotransmitterfunctions. Traditionally, motivated behaviors have been divided intoappetitive and consummatory components. Activities aimed at obtainingreward and satisfaction belong to the appetitive component. Thefundamental appetitive motivational process is an intrinsic brainfunction and is especially related to the predictive value of stimulifor reward. Processing of motivationally relevant information (i.e.,stimuli predicting reward) causes an increase in activity of themeso-accumbens dopaminergic (DA) system (i.e., DA neurons of the ventraltegmental area (VTA) innervating the nucleus accumbens (NAS)), acomponent of the mesolimbic dopamine system. The activity of this systemis increased during flexible approach behavior when anticipating rewardrelated to copulation.² Increasing activity in these dopaminergicpathways facilitates sexual motivation, in particular, anticipatorysexual behavior.³

Aripiprazol is, among others, an example of a drug that influencesdopaminergic pathways and that may be used in combination withtestosterone, or an analogue thereof, and a PDE5 inhibitor to affectsexual motivation and behavior. Aripiprazol is a high-affinity partialagonist of the dopamine D2 receptor and serotonin 5-HT1a receptor andantagonist of the 5-HT2a receptor. Aripiprazol is described as adopamine system stabilizer that is due to its partial agonistic actionsat the D2 receptor, especially the presynaptic D2 receptors, for whichit has higher affinity. Stimulation of autoreceptors located on dopaminenerve terminals results in an inhibition of dopamine synthesis andrelease. Thus, in a low dopaminergic state of the meso-accumbens DAsystem, aripiprazol would antagonize presynaptic D2 receptors, freeingthe NAS-projecting DA nuclei in the VTA from autoinhibition.

The medial prefrontal cortex (mPFC) mediates behavioral inhibition.Dopamine in the mPFC plays an important role in behavioral inhibition.Illustrative of mPFC-DA's inhibitory role is the inhibition of themeso-accumbens DA system, high extracellular concentrations of mPFC-DAinhibiting meso-accumbal DA activity, and low extracellularconcentrations of mPFC-DA activating meso-accumbal DA activity throughdisinhibition. It is, therefore, conceivable that a dopaminergic role inFSD is not restricted to meso-accumbal DA, but extendable to mPFC-DA,where symptoms of FSD are enhanced with high activity of mPFC-DA, albeitvia inhibition of accumbal DA or via inhibition of other cognitive oremotional factors involved in FSAD. The partial agonistic action ofaripiprazol will then have a positive effect on alleviation of FSD(symptomatology) through agonism of presynaptic D2 receptors in themPFC, thereby inhibiting DA release in this area.

Anticipating sexual reward will produce arousal of the genitalia, inwhich at least three key neurotransmitters are involved: acetylcholine,norepinephrine and nitric oxide. Acetylcholine and nitric oxide bothpromote erections in men and lubrication and swelling in women.Norepinephrine inhibits erections in men and lubrication and swelling inwomen. Orgasm, the consummatory phase of human sexual response, isfacilitated by descending spinal noradrenergic fibers and innervation ofthe genitalia, and inhibited by descending spinal serotonergic fibers.

Testosterone in Women

In many mammalian species, female sex steroids are necessary for theexpression of female sexual behavior. As a result, the capability forcopulation in these animals is limited to the period of ovulation.^(4,5)Higher primates, like humans, show sexual intercourse outside theperiovulatory period. For these animals, it has been suggested thattestosterone is involved in female sexual behavior.⁶ The disappearanceof testosterone following ovariectomy and adrenalectomy is accompaniedby a complete loss of libido,^(7,8) while substitution of this steroidmaintains sexual desire and fantasies after surgical menopause.⁹

Testosterone, Exposure to Sexual Cues and Vaginal Arousal in NormalWomen

An important aspect of sexual motivation is physiological sexualresponding. Measured as an increase in vaginal vasocongestion elicitedby sexual stimuli, this responding is considered to be preparatory forcopulatory behavior.¹⁰ In hypogonadotropic hypogonadal females, we foundthat substitution with testosterone undecanoate 40 mg orally per dayduring an eight-week period enhanced vaginal responsiveness.¹⁰ Thiseffect was not found in another group of hypogonadotropic hypogonadalpatients (unpublished data). In both studies, subjects receivedtestosterone each morning, but patients in the first experiment weretested in the afternoon and patients in the second experiment in themorning. The different outcomes on physiological responding betweenthese experiments may be caused by a time-dependent effect oftestosterone on vaginal arousal.

In a third experiment, we examined whether administration of a singledosage of testosterone sublingually, as compared with a placebo,increases vasocongestion during presentation of visual erotic stimuli.¹¹On treatment days, we exposed eight sexually functional women withintervals of an hour and a half to six erotic films depictingintercourse. The intake of testosterone caused a sharp increase inplasma levels of testosterone of short duration. About three to four anda half hours after this testosterone peak, we found a striking increasein vaginal responsiveness when the subjects were exposed to the visualsexual stimuli (see also FIG. 1). These findings demonstrate a time lagin the effect of sublingually administered testosterone on genitalarousal in sexually functional women.

The results of the above-mentioned studies demonstrate that testosteroneis involved in female sexual motivation in a time-dependent fashion. Theinfluence of sex steroids on sexual behavior might be explained by asteroid-responsive neural network, a highly interconnected group of sexhormone receptor-containing neurons in the brain.¹² This network is nota closed circuit, but serves reproductive aims by functioning as anintegrating and activating center between external sensory cues,hormonal processes and reproductive behavior. This is partlyaccomplished by selective filtering of sensory input and amplificationof signals that may facilitate sexual behavior. We assume that anincrease in vaginal vasocongestion induced by sexual stimuli ispreparatory for copulatory behavior. Visual exposure to sexualintercourse between members of the species of the onlooker is a potentreleasing stimulus for such a preparatory motivational response. Bothmen and women have a marked capacity to respond to erotic films with agenital response.¹³ The increased motivational sensitivity for sexualcues induced by testosterone is presumably the result from an alteredbrain state, in which dopaminergic, serotonergic and noradrenergicpathways are involved. This altered state might also be sensitive tointernal cues as evoked by sexual fantasy.

In another experiment, we demonstrated that attention directed atalterations in genital arousal produced concordance betweenphysiological and subjective indices of sexual arousal.¹⁴ There arereciprocal relationships between sexual desire, sexual arousal and theability and potency of orgasm. A reduced sexual desire will affectsexual arousal and visa versa; both indices of sexual function mightinfluence orgasm potency and visa versa.

Phosphodiesterase 5 Inhibitors and Sexual Arousal

In several studies, it has been shown that selective type 5phosphodiesterase (PDE5) inhibitors improve erectile function in menwith erectile dysfunction, on average close to normal function.¹⁵ In thepenis, nitric oxide (NO) released from nerves and endothelium inducesproduction of cyclic guanosine monophosphate (cGMP). cGMP is a keymechanism in relaxing smooth muscle, necessary for the induction of anerection. This nucleotide is hydrolyzed by the phosphodiesterases, fromwhich the main activity in the corpora cavernosa is due to PDE5.Therefore, during sexual stimulation, the action of NO/cGMP on erectilefunction will be enhanced by PDE5 inhibitors.¹⁶

The genitalia of both sexes have common embryological origins. Recently,it has been shown that the clitoris consists of an erectile tissuecomplex, which embeds the anterior vaginal wall. Clitoral erection andthe anterior wall of the vagina are highly involved in female sexualarousal and response. It has recently been shown that sildenafil, a PDE5inhibitor, improves sexual performance in sexually functional women.

Although in both men and women similar specialized vascular mechanismare involved in the genital response, an increase in Vaginal PulseAmplitude (VPA) cannot be considered to be the equivalent of anerection. A necessary but insufficient condition for an erection isdilatation of arteries and resulting increased blood inflow. In thepenis, there are corpora (corpus cavernosa (two) and corpus spongiosum(one)) containing small irregular compartments (vascular spaces). Thesmooth muscles in the cavernous sinusoidal walls are normally tonicallyconstricted under the control of an active sympathetic (adrenergic)tone. Relaxation of cavernous smooth muscle of the corpora results infilling and enlargement of the compartments with blood, which will beaccompanied by an erection.

Although the precise mechanisms are unknown, sympathetic innervationsand Nitric Oxide are believed to be both principal mediators inrelaxation of the corporeal smooth muscles. In the penis, sympatheticinnervations of the blood vessels are sparse, while the smooth musclesare richly innervated by this system. In contrast, the blood vessels ofthe penis are richly innervated by the parasympathetic system, whileinnervations of the smooth muscles by this system are sparse.Consequently, there are relatively independent effects of these twoparts of the peripheral nervous system on processes involved in theoccurrence of an erection. Initiation of dilatation of the penilearteries and subsequent increase of blood flow to the cavernous tissueis regulated by the parasympathetic nervous system (initiation of anincrease in this cholinergic activity depends on signals by the brain).However, without relaxation of the smooth muscles, there will be noerection.

Reduction of the sympathetic tone and consequent relaxation of smoothmuscles appears to be a relative independent prerequisite for theinitiation of an erection. Thus, penile erection occurs in response toincreased activity of the sacral parasympathetic innervations and adecreased activity of sympathetic pathways. In the penis, nitric oxide(NO) released from nerves and endothelium induces production of cyclicguanosine monophosphate (cGMP). cGMP is a key mechanism in relaxingsmooth muscle, necessary for the induction of an erection. Theproduction and release of NO might be influenced by a decrease inactivity of the sympathetic branch.

Brain Activity Mediates the Influence of Psychosocial Circumstances onSexual Behavior and Sexual Feelings via Inhibitory and ExcitatoryRegulating Mechanism

The prefrontal cortices of the human brain are crucial for cognitivefunctions involved in planning, execution and control of behavior. Animportant aspect of these cognitive functions is inhibition of limbicsystem-induced emotional responses, such as sexual behavior.Psychological processes are also involved in three distinct(transitional and overlapping) phases of the human sexual response, aswell as disturbances in these phases leading to low sexual desire,sexual arousal problems and hampered orgasm. Thus, an increase inactivity of the prefrontal cortex associated with inhibition mightreduce sexual desire, sexual arousal and orgasm capacity.

Activity in the prefrontal cortices is also involved in the regulationof the sympathetic and parasympathetic branches of the peripheralnervous system. An increase in prefrontal activity is accompanied by adecrease in parasympathetic activity and an increase in sympatheticactivity. Alterations in these branches occur asymmetrically. Moreover,the sympathetic branch of the peripheral nervous system seems to be moresensitive to psychogenic induced alterations. Thus, psychologicalprocesses controlled by the prefrontal cortices appear to be directlyinvolved in the physiological mechanisms regulating induction of anerection. It might be assumed that the same physiological mechanisms areregulating the different components of the female sexual response (i.e.,parasympathetic innervations are regulatory for the VPA and sympatheticinnervations are, together with Nitric oxide, responsible for swellingand lubrication).

In a recently conducted experiment (not published) in healthy sexuallyfunctional women, we used a delayed measurement design in which subjectsingested one dosage of testosterone (0.5 mg sublingual) or placebo andafter four hours, underwent fMRI while watching neutral and eroticvideos. Hereafter, subjects were conducted to a laboratory where theirVPA in response to neutral and erotic videos was measured. As expected,in the placebo condition, we found activation of cortical andsubcortical structures and deactivation of dorsal prefrontal areascomparable to other imaging studies on brain activation in response toerotic stimulation. Because of testosterone's delayed effect onenhancing VPA, we expected enhancement of these “erotic” structures inthis fMRI study. However, the opposite is true. Women on testosteroneshow a decreased activation of all brain structures during eroticexposure implicated in the normal sexual response. Furthermore, twostructures showed a very significant increase: the septum, whichfunctions as a restrainer of emotional overshoot, and the leftdorsolateral PFC, which functions as inhibitor of automated/reflexiveresponses. As stated, usually deactivation of dorsal prefrontal areas isobserved.

Depending on circumstances and individual differences, testosterone canproduce effects that deviate from the expectations one would have giventhe functional role of testosterone in the regulation of sexualbehavior. This inhibition of the centrally regulated autonomous sexualresponse was also apparent in the relative change in the VPA. Contraryto the expectations, the VPA was smaller in the testosterone conditionas compared with the placebo, and probably the result of a continuinginhibition mechanism induced during the fMRI procedure.

The Synergistic Effect of a Combination of Testosterone and PDE5Inhibitor on Vaginal Arousal in Women Suffering from Female SexualDisorder

In a recently conducted experiment (see experimental part) in womensuffering from Female Sexual Dysfunction, we found at different timeintervals no effect from one dosage of testosterone, nor from a PDE5inhibitor as compared with a placebo, on vaginal arousability, nor onsexual desire and genital sensations. In this experiment we found,however, that one dosage of testosterone (0.5 mg sublingual), combinedwith the administration of a PDE5 inhibitor (dosed in a way such that Tmax arises about 3.5 to 5.5 hours after the free testosterone peak),four hours after the plasma testosterone peak, caused a significantlyhigher Vaginal Pulse Amplitude during exposure to erotic stimuli. Thiseffect was less pronounced in a subgroup of women who were sexuallyabused during their childhood. We found no effect on sexual desire andon subjective sexual arousal. Treatment with testosterone, as well asthe combination of testosterone and a PDE5 inhibitor, caused an increasein attentional engagement (or withdrawal) for sexual cues, as comparedwith placebo and/or a PDE5 inhibitor. Attentional engagement is animportant function for normal human sexuality. Testosterone combinedwith a PDE5 inhibitor produced a statistically significant increase ingenital arousal.

In one of its embodiments, the invention provides the use of acombination of a PDE5 inhibitor and testosterone, or an analoguethereof, in the preparation of a medicament for the treatment of femalesexual dysfunction.

Testosterone is also known under the chemical name17-β-hydroxyandrost-4-en-3-one which can be obtained in various ways: itmay be isolated and purified from nature or synthetically produced byany manner. The term “or an analogue thereof” includes any usefulmetabolite or precursor of testosterone, for example, the metabolitedihydrotestosterone. It is clear to the skilled person that if ametabolite or precursor of testosterone is used, the time point foradministration of a PDE5 inhibitor probably needs to be adapted. If, forexample, dihydrotestosterone is used, the time of administration of thePDE5 inhibitor lies approximately half an hour earlier (as this is theapproximate time it takes for excess testosterone to be converted todihydrotestosterone). The amount of PDE5 inhibitors is still expandingand non-limiting examples are the following: GF-196960/IC351(tadalafil), Bay-38-9456 (vardenafil), UK-103320 (Sildenafil), E-4021,E-8010, E-4010, AW-12-217 (zaprinast), AWD 12-210, UK-343,664,UK-369003, UK-357903, BMS-341400, BMS-223131, FR226807, FR-229934,EMR-6203, Sch-51866, IC485, TA-1790, DA-8159, NCX-911 or KS-505a. Otherexamples can be found in WO 96/26940.

Preferably, the PDE5 inhibitor is provided at least three hours afterthe administration of the testosterone, even more preferably, such thatC_(max) arises about 3.5 to 5.5 hours after the plasma free testosteronepeak. As already described above and depending on the formulation, thePDE5 inhibitor can also be given at the same time testosterone isadministered.

Conditioning of Positive Associations Between Different Modalities ofthe Sexual Response

Treatments with a dosage of testosterone combined with a PDE5 inhibitorproduce alterations in brain and bodily functions that will makelearning of positive associations between sexual stimuli, genitalarousal and subjective experience possible. Moreover, the treatment ofFSD with a combination of testosterone and a PDE5 inhibitor ispreferably augmented by an “approach induction” treatment. To create amore permanent psychological change, the central and bodily processesactivated by testosterone and a PDE5 inhibitor under sexually relevantstimulation need to be perceived and need to become associated with apositive hedonic tone or with activation of the behavioral approachsystem. The perception of bodily reactions by focusing attention ongenital arousal is made possible by testosterone (whereby the genitalarousal is synergistically enhanced by the PDE5 inhibitor) and can beemphasized by verbal instructions. A positive hedonic tone cannot betaken for granted in the population of FSD patients. In order to achievea positive tone, patients can be exposed to positive stimuli during theeffective phase of the drugs (that is, at least three hours aftertestosterone intake). These positively motivated stimuli consist ofpictures of happy faces of persons of the patient's sexually preferredgender, possibly including the face of the partner. The pictures of thefaces are presented subliminally, so that in an unobtrusive way thebehavioral approach system becomes activated.

The treatment of FSD might consist of creating a situation in which thepatient learns to associate genital arousal with a positive hedonic toneor activation of the behavioral approach system. This requiresinducement of genital arousal (by sexual stimuli and a PDE5 inhibitor),sustained attention to sexual stimuli and to genital arousal (madepossible by testosterone) and activation of the behavioral approachsystem (by subliminal presentation of pictures of happy faces).

The invention further provides a method for treating a female sufferingfrom female sexual dysfunction by providing to the female a combinationof a PDE5 inhibitor and testosterone, or an analogue thereof.

The invention will be explained in more detail in the following,non-limiting examples.

EXAMPLES

Participants

Fourteen women with a heterosexual orientation (mean age: 40.6 years;sd: 10.4; premenopausal n=8, postmenopausal n=6) who have beenexperiencing FSD (i.e., low sexual desire, low sexual arousal ordecreased orgasm potency) for at least six months prior to study entry,participated in this study. Subjects in fertile age used contraceptives(IUDs, sterilization, oral contraceptives except contraceptivescontaining anti-androgens). A pregnancy test was part of the procedure.Subjects were interviewed and examined by a gynecologist to excludepregnancy or breast feeding, vaginal infections, major operations to thevagina and/or vulva or unexplained gynecological complaints.Participants did not have a history of endocrinological, neurological orpsychiatric treatment. Cardiovascular condition was tested and ECG waschecked for significant abnormalities. Standard blood chemistry andhematology tests were performed. Participants did not abuse drugs andwere required not to use alcohol or psychoactive drugs the eveningbefore and the day of experimentation. Subjects could not makeappointments during their period of menstruation.

Procedures

This study was approved by the Dutch medical-ethical committee(STEGMETC). The experimental trials were preceded by a screening visit.At this screening visit, subjects were interviewed by apsychologist/gynecologist to diagnose for FSD and to determineeligibility for study participation. Weight, height, blood pressure(supine and standing), heart rate, respiration rate, and bodytemperature were measured. A supine, 12-lead ECG was recorded andexamined by a physician (and, when necessary, a cardiologist). Agynecological examination and urine pregnancy test were performed.Cultures were taken to exclude Chlamydia or Gonococcus infections.

Eligible subjects underwent a familiarization trial following thescreening. A trained female experimenter familiarized the subject withstudy requirements and procedures. This included the use of the vaginalphotoplethysmograph (a tampon-shaped device). The subjects viewed aneutral film fragment of five minutes followed by an erotic filmfragment of five minutes. Hereafter, they practiced on a shortenedversion of the emotional Stroop task.

For the experimental trials, we used a double-blind, randomly assignedplacebo controlled cross-over design with four drug conditions:

-   -   1) Placebo    -   2) PDE5 inhibitor    -   3) Testosterone (0.5 mg sublingual)    -   4) testosterone+PDE5 inhibitor (the two compounds were given at        the same time, but the PDE5 inhibitor was dosed/formulated such        that the effect of the testosterone and the effect of the PDE5        inhibitor at least partly overlapped).

Each subject underwent the four different drug treatments (i.e., PDE5inhibitor, testosterone, PDE5 inhibitor+testosterone and placebo) atfour separate experimental days. The four experimental days wereseparated by at least a three-day period.

During each day of drug manipulation, subjects underwent the followingmeasurements:

-   -   −1:00 hours: Emotional Stroop Task    -   −0:15 hours: Trial 1    -   0:00 hours: Intake drugs    -   0:05 hours: Trial 2    -   2:45 hours: Trial 3    -   4:15 hours: Trial 4

Each day started with a physical examination (measurements of vitalsigns, blood pressure, heart rate, temperature and respiration rate).Blood samples (8 ml) were taken for hormone analyses. Subjects then wereseated in the sound attenuated, dimly lit experimental room. In order tomake this room less “sterile,” geographic posters were hung on the wallsand an air perfumer was placed behind the subjects. The subjects thenexecuted the emotional Stroop task (15 minutes). The experimenterbrought the vaginal probe and the subject was left alone in the room toinsert the probe. The subject was instructed to sit as quietly aspossible while viewing the film fragments. A ten-minute neutral fragmentwas followed by a five-minute erotic film fragment. After these baselinemeasurements, the subjects took medication: testosterone (0.5 mg orplacebo) sublingually, with cyclodextrins as carrier and Vardenafil (10mg or placebo) hidden in a capsule. The medication was followed byanother set of neutral (five minutes) and erotic (five minutes) filmfragments. The subjects removed the vaginal probe and were taken to awaiting room for a pause of two hours. During this pause, they couldconsume their lunch. Unlimited coffee and tea were available. This pausewas followed by a third set of neutral (five minutes) and erotic (fiveminutes) film fragments.

Again subjects had to wait for two hours so that the last VPAmeasurement was taken four hours after medication. This last film-trial(neutral, erotic film fragments) was followed by a second presentationof the emotional Stroop. The experimental day ended with a shortphysical examination including drawing of a blood sample (8 ml forhormone analyses) and collection of AE and SAE.

FIG. 2 shows the effect of the pharmaceutical preparation according tothe invention (P<0.035) as compared with a placebo/PDE5 inhibitor on VPAin women suffering from FSD.

FIG. 3 shows the effect of the pharmaceutical preparation according tothe invention (P<0.04) as compared with a placebo, a PDE5 inhibitor andtestosterone in women suffering from FSD.

Emotional Stroop Task

An unmasked and a masked version of the Emotional Stroop Task comparingcolor-naming latencies on neutral and erotic words were used. In boththe unmasked and the masked condition, eight erotic and neutral wordsare presented in different colors (i.e., red, green, blue and yellow).An extra set of stimuli consisting of letter strings was used forpractice trials. Subjects were instructed to ignore the content of thewords and to name the color of the words as quickly as possible (in themasked condition, the color of the mask has to be named). Each trialconsisted of a fixation point which is shown for 750 ms, followed by thetarget stimulus (the colored neutral or erotic word). In the maskedcondition, the word picture is presented for about 24 ms and then maskedby randomly cut, reassembled letters in the same color. A microphoneconnected to a voice-level detector was placed in front of the subject.Initiation of vocal response was registered by the computer's clock andterminated the target presentation (with a no-response maximum of 3000ms). Thirty-two neutral words and thirty-two erotic words were presentedblocked. The same words were used for each test, however, the sequenceof words and colors differed all eight times this task was used.

Results

VPA Measurements

During the testosterone condition, one subject became nauseated watchingthe erotic film and decided not to participate further that day. For thepsychophysiological evaluation, the VPA (Vaginal Pulse Amplitude) isused. The VPA reflects phasic changes in the blood volume correspondingwith each heartbeat; higher levels indicate higher levels of blood flow.The dependent variable used is the amplitude of the pulse wave. Beforethe mean VPA was calculated, the raw signal (sample rate was 20 Hz) wasdigitally bandpass filtered with a Butterworth filter (−3 dB cutofffrequency range 0.7-1.5 Hz; 40 dB down/octave). Movement artifacts weredetected by visual inspection of the signal and removed manually.Hereafter, the amplitude was measured as the distance between the topand bottom of a pulse wave. The mean VPA was calculated as the averageof these amplitudes across 30-second periods.

The magnitude of the VPA response also depends on the exact placement ofthe probe. In order to compare conditions, it is not meaningful toexamine the absolute mean amplitude values in the neutral or eroticconditions. Instead, we use the mean change in amplitude from neutral toerotic film fragment divided by mean score in the neutral conditionduring each film trial as endpoint in the analyses.

The resulting difference scores were subjected to a 2 Steroid(testosterone yes/no)×2 PDE5 inhibitor (Vardenafil yes/no)×2before-after (trial 1 versus trial 4) repeated measures ANOVA. Aninteraction effect was found for the steroid condition over trials(F(1.12)=5.75; p<0.04), implicating that the increase from before toafter measurement was significantly higher for drug conditionscontaining the steroid (testosterone+placebo andtestosterone+Vardenafil) than for the drug conditions without thesteroid (placebo+placebo and placebo+Vardenafil). Further analyses ofthe before medication (trial 1) versus after medication (trial 4)responses showed that the increase in VPA between neutral and eroticfilms was insignificant in the placebo condition and in the Vardenfil ortestosterone condition (see also FIG. 4). Only the combined treatmentcondition (Vardenfil+testosterone) lead to a statistically significantincrease in VPA before compared with after medication (F(1.12)=3.229;p=0.007).

We hypothesized the effects of sexual stimulation and medication ongenital responses to be associated with changes in central mechanisms.To test for associations with changes in attentional processes, we usedthe StroopRT as the measurement point. StroopRT is the difference inmean reaction times on color naming of the neutral and the sexual words.

During visual inspection of the data, we found two subgroups within oursample: a group of women who were sexually abused during childhood and agroup of women who did not report such abuse. We decided to includesexual abuse during childhood as a between subjects variable in theanalyses.

In the unmasked condition, no statistically significant results werefound. Analyses reported heretofore reflect results in the maskedcondition of the Stroop task. The MANOVA revealed a significantinteraction between Steroid, Trial and Sexual-abuse-in-childhood(F(2.10)=13.6; p=0.001). Within-subjects contrasts confirmed significanteffects for both VPA (F(1.11)=10.97; p=0.007) and StroopRT(F(1.11)=5.85; p=0.034) (FIG. 5).

Subjects who have been sexually abused during childhood show anattenuated VPA to erotic stimuli (before medication increase in VPAduring erotic film relative to neutral film is <60%, compared to >90%increase for not-abused subjects) and no increase over trials with orwithout testosterone. However, these subjects are not insensitive totestosterone. Under influence of testosterone, they develop anattentional bias away from sexual stimuli (StroopRT increases).Not-abused subjects show an opposite pattern: their attention for sexualstimuli increases (StroopRT decreases) and parallel their VPA responseto the erotic film increases in the testosterone versus no-testosteroneconditions. Univariate analyses: VPA: 2 Steroid (testosterone yes/no)×2Trial (before—after medication) ANOVA with VPA as within subjectsdependent measure and sexual abuse as between subjects factor:F(2.10)=5.9; p=<0.035; StroopRT: 2 Steroid (testosterone yes/no)×2 Trial(before—after) ANOVA with StroopRT as dependent measure and sexual abuseas between subjects factor: F(2.10)=4.2; p<0.07.

In this group of women suffering from FSD, Vardenafil did not lead to asignificant difference in VPA under condition of sexual stimulationcompared to placebo. Apparently, peripheral manipulations are notsufficient and central mechanisms need to be influenced. We show thattestosterone is such a centrally acting influence on sexual mechanisms.However, for our subjects, testosterone alone was insufficient for asignificant increase in VPA compared to placebo. Only the combination oftestosterone and Vardenafil lead to a significant increase in VPAcompared with placebo.

We found that we could distinguish subgroups of subjects. For a subgroupof sexually abused women, testosterone had an effect on attention forsexual stimuli. When these subjects had a greater attention for sexualstimuli, their genital response did not increase. Both healthy women andwomen with FSD who are not abused respond with a genital reaction whentheir attention is directed to sexual stimuli. We showed in this studythat for patients without a history of abuse, the combination oftestosterone and a PDE5 inhibitor is beneficial in that it increasesboth resources allocated to the processing of sexual stimuli, as well astheir genital response to these sexual stimuli.

The observed effect was less pronounced in a subgroup of women who weresexually abused during their childhood. For these women, thetestosterone, or an analogue thereof, and a PDE5 inhibitor is optionallycomplemented with psychotherapeutic intervention.

In a small study (N=4), we further investigated the efficacy ofsubliminal presentation of faces of happy men during erotic film clipsin four healthy women, following administration of the testosterone andPDE5 inhibitor combination. Women reported increased subjective sexualarousal in the subliminal happy male face presentation condition, incomparison to erotic film excerpts only condition.

REFERENCES

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1. A method of treating a sexual disorder in a female, said methodcomprising: administering to a female suffering from said sexualdisorder a combination of a PDE5-inhibitor and testosterone or ananalogue thereof, so as to treat the sexual disorder.
 2. A method oftreating a patient suffering from sexual dysfunction, said methodcomprising: administering to the patient testosterone or an analoguethereof to provide a peak level in the patient's blood circulation offree testosterone or an analogue thereof of 0.010 nmol/l, so as to treatthe patient suffering from the sexual dysfunction.
 3. The methodaccording to claim 1, wherein the testosterone or an analogue thereof isprovided in the form of a sublingual formulation.
 4. The methodaccording to claim 3, wherein said sublingual formulation furthercomprises cyclodextrin.
 5. A pharmaceutical formulation comprising:testosterone or an analogue thereof, wherein said formulation isdesigned to release essentially all testosterone or an analogue thereofwithin a time interval of 60 seconds upon administration.
 6. Thepharmaceutical formulation of claim 5, which is designed for sublingualadministration.
 7. The pharmaceutical formulation of claim 6, furthercomprising a cyclodextrin.
 8. The pharmaceutical formulation of claim 7,wherein said cyclodextrin comprises hydroxypropyl-beta cyclodextrin. 9.A kit of parts comprising: a first pharmaceutical composition comprisingtestosterone or an analogue thereof, wherein said first pharmaceuticalcomposition is designed to release all testosterone or analogue thereofessentially immediately at the target site, and a second pharmaceuticalcomposition comprising a PDE5-inhibitor.
 10. The kit of parts of claim9, further comprising: instructions to administer the pharmaceuticalcomposition from 3.5 to 5.5 hours prior to sexual activity, and toadminister the second pharmaceutical composition from one (1) to two (2)hours prior to sexual activity.
 11. The kit of parts of claim 9, whereinthe first pharmaceutical composition comprises from 0.3 mg testosteroneto 2.5 mg testosterone.
 12. The kit of parts of claim 9, wherein thesecond pharmaceutical composition comprises from 5 mg to 20 mgvardenafil.
 13. The kit of parts of claim 9, wherein the secondpharmaceutical composition comprises at least 25 mg and at most 100 mgsildenafil.
 14. The kit of parts of claim 9, wherein the secondpharmaceutical composition comprises from 5 mg to 20 mg tadalafil. 15.The kit of parts of claim 9, further comprising: a compound competingwith testosterone or an analogue thereof for SHBG binding.
 16. The kitof parts of claim 9, wherein said analogue of testosterone is aprecursor or a metabolite of testosterone.
 17. The kit of parts of claim16, further comprising: instructions to administer the firstpharmaceutical composition 3.5-5.5 hours plus the time needed to converta precursor to testosterone, prior to sexual activity and to administerthe second pharmaceutical composition one (1) to two (2) hours prior tosexual activity.
 18. The kit of parts of claim 16, further comprising:instructions to administer said first pharmaceutical composition 3.5-5.5hours minus the metabolization time of testosterone to the metabolite,prior to sexual activity and to administer the second pharmaceuticalcomposition one (1) to two (2) hours prior to sexual activity.
 19. Thekit of parts of claim 9, further comprising: means for cognitiveinterventions directing attention to genital arousal.
 20. The kit ofparts of claim 9, further comprising: means for cognitive interventionsdirecting attention to genital arousal during appropriate conditions forsufficient sexual stimulation.
 21. The kit of parts of claim 19, whereinsaid means for cognitive interventions inducing activation of thebehavioral approach system or a positive hedonic tone by subliminalexposure to pictures of happy faces of persons of the patient's sexuallypreferred gender.
 22. The kit of parts of claim 19 wherein said means ispart of a website.
 23. The kit of parts of claim 9, further comprising:an agonist of the dopamine pathway.
 24. The kit of parts of claim 9,further comprising: inhibition of adrenergic tone in the central nervoussystem.
 25. The method according to claim 2, wherein the testosterone oran analogue thereof is provided in the form of a sublingual formulation.26. The method according to claim 25, wherein said sublingualformulation further comprises cyclodextrin.